Culture system and method for maintenance and proliferation of undifferentiated human embryonic stem cells

ABSTRACT

The present invention discloses an improved culture system for proliferative and undifferentiated growth of human embryonic stem cells, comprising an extracellular matrix prepared from feeder cells and a conditioned medium preconditioned by feeder cells. This invention also relates to a culture method utilizing the foregoing culture system.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the field of human embryonic stem (HES)cell culture, in particular undifferentiated HES cell culture, and tomethods for maintenance and proliferation of such cells. Moreparticularly, the invention relates to maintenance of HES cells in asubstantially proliferative and undifferentiated status in a culturesystem without feeder layer.

2. Description of Related Art

Human embryonic stem (HES) cells are derived from the inner cell mass ofblastocyst. Nowadays, most HES cells can only be maintained in culturein an undifferentiated state when grown on inactivated feeder cells.

Previous studies have found that the important factors in feeder cellsthat support the growth and proliferation of ES cells include growthfactors secreted by feeder cells in the culture medium and extracellularmatrix (ECM) constructed by the feeder cells. The feeder cells can be aprimary mouse embryonic fibroblast (PMEF), a mouse embryonic fibroblastcell line (MEF), a murine fetal fibroblast (MFF), a human embryonicfibroblast (HEF), a human fetal muscle cell (HFM), a human fetal skincell (HFS), a human adult skin cell, a human foreskin fibroblast (HFF),a human adult fallopian tubal epithelial cell (HAFT) and a human marrowstromal cells (hMSCs) (WO 03/02944, WO 03/014313, J. H. Park et al.,Biol Reprod., 69: 2007-2017, 2003, M. Amit et al., Biol Reprod., 68 (6):2150-2156, 2003, Outi Hovattal et al., Hum. Reprod., 18 (7): 1404-1409,2003, Richards, M. et. Al, Nat Biotechnol., 20 (9): 933-936, 2002, JamesA. et al., Science, 282 (6): 1145-1147, 1998 and Linzhao Cheng et al.,Stem Cells, 21: 131-142, 2003).

The extracellular matrix (ECM) is not merely a passive structure. In thepast few years, it has emerged that the matrix is a dynamic action zonethat functions to instruct cellular phenotype. ECM proteins interactdirectly with cell surface receptors to initiate signal transductionpathways and to modulate those triggered by growth factors. ECM alsocontrols the activity and presentation of a wide range of growthfactors. Thus modulation of the ECM, by remodelling its structure andactivity, has profound effects on its function and the consequentbehaviour of cells residing on or within it. At present, the interactingmechanisms of the ECM in establishment and maintenance of a ES cellculture is not known. Possible roles for the ECM include the provisionof ECM components that provide attachment sites for the ES cells,trigger signaling for cell renewal.

The feeder layer dependent culture system in scaling up and impedes themass production and clinical application of HES cells. There are someproblems in a feeder layer dependent culture system: (1) the potentialrisks of transmission of pathogens from the animal feeder cells to theHES cells and the fact that the current system of propagation(human/animal or human/human co-culture) has been construed as axenotransplant, (2) feeder cells come mainly from primary cells, whileprimary cells from different batches offer different effect as feedercells, rendering the quality control of the cultured HES cells moredifficult; (3) the limited sources and numbers of feeder cells hamperthe mass production and applications of HES cells. Therefore, the methodfor the maintenance and proliferation of undifferentiated HES cellswithout feeder cells is critical for mass product and clinicalapplication of HES cells. (U.S. 2003/143736).

Xu et al. (Nat. Biotechnol., 19 (10): 971-974, 2001. WO 03/020920 andU.S. 2003/0017589) were the first to successfully maintainundifferentiated HES cells in a feeder-free culture system. In thissystem, HES cells are cultured on Matrigel from the Engelbreth HolmSwarm (EHS) sarcoma or laminin in medium conditioned by MEF. However,such synthetic matrices and defined-matrix marcromolecules are notsufficient to mimic the more complex cell-martix interactions providedby feeder cells. A study has also indicated that this culture system isonly suitable for certain HES cell lines (e.g. H1 and H9), butunsustainable for other HES cell lines (Outi Hovattal et al. Hum.Reprod., 18 (7): 1404-1409, 2003).

Accordingly, it is an object of the present invention to provide analternative feeder-free culture system to overcome some of thedisadvantages of the prior art.

SUMMARY OF THE INVENTION

The present invention provides methods and culture system for culturingundifferentiated HES cells. The methods and culture system describedherein provide improved culture conditions that allow the maintenanceand proliferation of HES cells in a substantially undifferentiatedstate.

In one aspect, the present invention provides a cell culture system forgrowing HES cells in a substantially undifferentiated state. The cellculture system of the invention comprises ECM as culture matrix andconditioned medium.

The feeder cells to be used in the present invention can be, forexample, but not limited to primary mouse embryonic fibroblasts (PMEF),a mouse embryonic fibroblast cell line (MEF), murine fetal fibroblasts(MFF), human embryonic fibroblasts (HEF), human fetal muscle cells(HFM), human fetal skin cells (HFS), human adult skin cells, humanforeskin fibroblasts (HFF), human adult fallopian tubal epithelial cells(HAFT) and human marrow stromal cells (hMSCs).

In another aspect, the present invention provides a culture method forgrowing HES cells in a substantially undifferentiated state, comprisingculturing undifferentiated HES cells in a feeder-free culture system ofthe invention. The HES cells cultured in such culture system may bemaintained in substantially proliferative and undifferentiated state forat least five passages.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the flow chart of culturing HES cells in the feeder-freeculture system according to this invention.

FIG. 2A shows the morphology of primary mouse embryonic fibroblasts(PMEF) (300×).

FIG. 2B shows the structure of ECM of PMEF (300×).

FIG. 2C shows the macrofibril bundles and network structure of ECM ofPMEF observed under scanning electron microscope (2000×).

FIG. 3A shows the morphology of human foreskin fibroblast (HFF) (300×).

FIG. 3B shows the structure of ECM of HFF (300×).

FIG. 4A shows the morphology of HES cells (HES3) cultured on ECMprepared from PMEF according to this invention (10×).

FIG. 4B shows the morphology of HES3 cells cultured on ECM prepared fromHFF according to this invention (10×).

FIG. 5A shows high alkaline phosphatase activity in HES3 cells culturedon ECM prepared from PMEF according to this invention (10×).

FIG. 5B shows high alkaline phosphatase activity in HES3 cells culturedon ECM prepared from HFF according to this invention (10×).

FIG. 6 shows the OCT-4 expression of HES3 cells cultured in thefeeder-free culture system according to this invention.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the present invention relates to a cell culture systemfor growing HES cells in a substantially undifferentiated state. Thisinvention is characterized by the removal of feeder cells, whileretaining the ECM structure and nutrients secreted by the feeder cells,hence developing a feeder-free culture system for HES cells, whichbreaks the bottleneck facing the large scale production and clinicalapplication of HES cells.

Specifically, this invention provides a culture system for growing HEScells, comprising a culture matrix consisting of ECM prepared fromfeeder cells and a conditioned medium being preconditioned by feedercells.

The aforesaid culture matrix provides substratum for cell attachmentduring culture and helps to maintain the HES cells in a substantiallyundifferentiated state.

The aforesaid feeder cells may be fibroblasts or other types of cells,which may be inactivated by large-dose radiation, such as y-ray, or bydrug, such as mitomycin C, so that the surviving cells lose thecapability to proliferate, but retain their physiological functions,such as metabolism and synthesis of growth factors. Specifically, thefeeder cell may be selected from the group consisting of primary mouseembryonic fibroblasts (PMEF), a mouse embryonic fibroblast cell line(MEF), murine fetal fibroblasts (MFF), human embryonic fibroblasts(HEF), human fetal muscle cells (HFM), human fetal skin cells (HFS),human adult skin cells, human foreskin fibroblasts (HFF), human adultfallopian tubal epithelial cells (HAFT) and human marrow stromal cells(hMSCs). In the preferred embodiments of the invention, the feeder cellsare derived from mouse embryonic fibroblasts (MEF) or human foreskinfibroblasts (HFF).

In the preferred embodiment of the invention, said HES cells are HES-3or HES-4 cell lines.

The preparation of ECM may be performed by those skilled in the art orrefer to R. Ian Freshney (2000) Culture of Animal Cells: A Manual ofBasic Technique, 4th Edition, Wiley-Liss, Inc. Basically, feeder cellswere treated with NaOH or trinitrotoluene (Triton) and followed byremoving intracellular substances, such as nucleus or organelles, so asto obtain the ECM as culture matrix. The removal of intracellularsubstances may be achieved by washing with buffered solution or water.

The aforesaid conditioned medium refers to culture medium in whichfeeder cells have been cultivated already for a period of time and thusis preconditioned by feeder cells. The preparation of conditioned mediummay be performed by those skilled in the art or refer to U.S. Pat. Nos.5,690,926, 2003/0008392, 2003/0073234, and 2002/0160509 as well asReubinoff B. E. et al, Nat. Biotechnol., 18 (4): 399-404, 2002. Themajor ingredients of a conditioned medium are typically amino acids,vitamins, carbohydrates, inorganic ions and some other auxiliarysubstances. Growth factors that promote cell growth or inhibitdifferentiation, e.g. leukemia inhibitory factor (ILF), fibroblastgrowth factor (FGF), stem cell factor (SCF),insulin-transferrin-selenium G supplement (ITS G supplement) may also beadded into the culture medium according to WO 03/020920, U.S.2003/0017589, U.S. Pat. No. 5690926, U.S. Pat. No. 5,453,357, Xu, C. etal., Nat. Biotechnol., 19 (10): 971-974, 2001, and Richards, M. et. Al,Nat Biotechnol., 20 (9): 933-936, 2002.

It is more specific that the preparation of the aforesaid conditionedmedium comprises of the steps of: (a) inactivating the feeder cells; (b)placing the cells obtained in step (a) in a culture solution; and (c)collecting the cell culture solution as conditioned medium. In anotheraspect, the present invention provides a culture method for growing HEScells in a substantially undifferentiated state, comprising culturingundifferentiated HES cells in the cell culture system of this inventionas shown in FIG. 1. The method comprises the following steps: obtainingHES cells; culturing the HES cells in the feeder-free culture systemdescribed above; and maintaining the HES cells in substantiallyproliferative and undifferentiated state, wherein HES cells in saidfeeder-free culture system may be maintained in substantiallyproliferative and undifferentiated state for at least five passages.

Definitions

The following terms will be defined as provided unless otherwise stated.All other terminology used herein will be defined with respect to itsusage in the particular art to which it pertains unless otherwise noted.

“Conditioned Medium”

Conditioned Medium as used for the purpose of describing the presentinvention refers to the medium in which feeder cells have beencultivated already for a period of time. The conditioned medium of thepresent invention can be used for cultivation of HES cells because itcontains many mediator substances, such as growth factors and cytokines,that were secreted by the feeder cells cultivated previously and canthus help to promote the growth of HES cells.

“Extracellular Matrix”

Extracellular Matrix or ECM or Defined Matrix occupies the space betweencells and establishes a complex network of different combinations ofcollagens, proteoglycans, hyaluronic acid, laminin, fibronectin, andmany other glycoproteins including proteolytic enzymes involved indegradation and remodeling of the ECM. ECM plays an important structuraland functional role in multicellular organisms and is more than ascaffold that fills extracellular spaces. Many of its components areengaged in processes mediating cell-to-cell interactions. In the presentinvention, ECM serves as culture matrix prepared from feeder cells so asto provide a support in a feeder-free culture environment for HES cells.

“Feeder Cells”

Feeder Cells as used for the purposes of describing the presentinvention refers to those used as a substratum on which other cells aregrown in a culture system. Feeder cells are usually adherentgrowth-arrested but viable and bioactive cells (primary cells orcontinuous cell lines) that have been incapacitated, for example byirradiation.

All other acronyms and abbreviations have the corresponding meaning aspublished in journals related to the arts of chemistry and biology.

The following examples are presented in order to more fully illustratethe preferred embodiments of the invention. They should in no way beconstrued, however, as limiting the broad scope of the invention. Whilethe invention is described and illustrated herein by references tovarious specific material, procedures and examples, it is understoodthat the invention is not restricted to the particular materialcombinations of material, and procedures selected for that purpose.Numerous variations of such details can be implied as will beappreciated by those skilled in the art.

EXAMPLE 1 Preparation of the Compositions of the Culture System

1. Preparation of Conditioned Medium

Conditioned medium for maintaining HES cells was prepared using thefollowing procedure. Primary mouse embryonic fibroblasts (PMEF) or humanforeskin fibroblast (from Animal Technology Research Institute, Taiwan)in the presence of a growth medium prepared from 10% fetal bovine serum(FBS, from HyClone) and 90% Dulbecco's Modified Eagle Medium (DMEM, fromGibco). When the cells reach confluence, mitomycin C was added toinactivate the fibroblasts. These fibroblasts were grown in the presenceof a growth medium prepared from 80% Dulbecco's modified eagle medium(DMEM, from Gibco), 20% fetal bovine serum (FBS, from Hyclone), andsupplemented with 1 mM β-mercaptoethanl (from Gibco), 1% non-essentialamino acids (from Gibco), 1% glutamine (from Gibco), and 1%insulin-transferrin-selenium G supplement (ITS G supplement, from Gibco)(refer to Richards, M. et al., Nat. Biotechnol., 20 (9): 933-936, 2002),in which fetal bovine serum may be substituted by serum replacement toobtain serum-free culture medium. The ES medium was collected andfilter-sterilized (0.2 micron filter). This medium was termed“conditioned ES medium”. The conditioned ES medium was used immediatelyor frozen at about −20° C. until needed. Based on the requirements ofthe cultured cells, the growth medium may contain other ingredientswithout limited to those discussed herein.

2. Preparation of Culture Matrix

Culture matrix for maintaining HES cells was prepared using thefollowing procedure. Primary Mouse embryonic fibroblasts (PMEF) or humanforeskin fibroblasts (from Animal Technology Research Institute, Taiwan)were grown to confluence in the presence of a growth medium preparedfrom 10% fetal bovine serum (FBS, from HyClone) and 90% Dulbecco'sModified Eagle Medium (DMEM, from Gibco). When the cells reachedconfluence, mitomycin C was added to inactivate the fibroblasts. Forharvesting the culture matrix, 0.05N NaOH or 0.1% trinitrotoluene (fromSigma) was used to break the cell membranes and followed by washing withpH 7.4 Dulbecco's Phosphate Buffered Saline (1X, from Gibco) to removeorganelle and nucleus so as to harvest the culture matrix.

EXAMPLE 2 Culturing HES Cells using the Culture System of this Invention

HES cells (HES-3 or HES-4 cell lines) were plated onto the culturematrix and incubated with conditioned medium mentioned above andcultured in a 5% CO₂ incubator under 37° C. The medium was changed every1-2 days. After 7 days of culture, HES cells were carried on subcultureof cells.

EXAMPLE 3 Observing the Culture Matrix of Culture System

FIG. 2A shows the morphology of primary mouse embryonic fibroblasts. Theculture matrix derived from primary mouse embryonic fibroblastsfollowing the steps in Example 1 is shown in FIG. 2B. When observedunder scanning electron microscope, the macrofibril bundles and networkstructure of said culture matrix are visible as shown in FIG. 2C.

FIG. 3A and FIG. 3B shows respectively the morphology of human foreskinfibroblast and the structure of culture matrix prepared from humanforeskin fibroblast according to the steps described above.

EXAMPLE 4 Analyzing the Undifferentiated State of HES Cells

The effect of the culture system herein may be further observed usingbiomarkers specifically expressed in undifferentiated HES cells, e.g.alkaline phosphatase activity, OCT-4, SSEA-3, SEA-4, TRA-1-60, andTRA-1-81 (refer to Thaomson J. A. et al, Science, 282 (6): 1145-1147,1998 or Reubinoff B. E. et al. Nat Biotechnol. 18 (4): 399-404, 2000) todetermine the undifferentiated level of ES cells.

This invention uses the expression of alkaline phosphatase activity andOCT-4 to evaluate the effect of the feeder-free culture system herein.The assay of alkaline phosphatase activity was employed according to theprotocols provided within the Alkaline phosphatase substrate kit (VectorLaboratories, Inc.). The assay of OCT-4 was carried out according to themethod described by Richards, M. et. Al, Nat Biotechnol., 20 (9):933-936, 2002. The results are presented as follows:

1. Observation of Cell Morphology:

FIG. 4A and FIG. 4B show the morphology of HES cells cultured in theculture system herein using primary mouse embryonic fibroblasts andhuman foreskin fibroblasts as culture matrix, respectively.

2. Assay for Alkaline Phosphatase Activity:

HES cells show high alkaline phosphatase activity beforedifferentiation, i.e., once they start to differentiate, they lose thealkaline phosphatase activity. Thus the differentiation status ofcultured HES cells can be learned from the this enzyme activity. Theassay results show that HES cells cultured in this culture system usingeither primary mouse embryonic fibroblasst or human foreskin fibroblastsas culture matrix express high alkaline phosphatase activity (in brightred color as a result of stain), indicating that they were in asubstantially undifferentiated state (FIG. 5A and FIG. 5B). The resultsalso showed that HES cells may be continuously subcultured for at leastfive passages and maintained the characteristics of substantialproliferation and undifferentiation in the feeder-free culture system ofthe present invention.

3. Assay for Transcription Factor OCT-4:

As shown in FIG. 6 which compares the expression of OCT-4 in HES cellscultured in various culture systems, lane 1 is feeder cells derived fromprimary mouse embryonic fibroblasts; lane 2 is feeder cells derived fromhuman foreskin fibroblasts; lane 3 is feeder-free culture systemprepared according to this invention; and lane 4 is a cell-free negativecontrol. Transcription factor OCT-4 can be observed in the ES cellscultured in the feeder-free culture system herein (lane 3), indicatingthat this culture system is able to maintain HES cells in substantiallyundifferentiated state. Besides, the expression level of OCT-4 infeeder-free culture system herein (lane 3) was comparable to that infeeder layer culture systems (lane 1 and lane 2).

The feeder-free culture system for ES cells presented herein offers thefollowing advantages: (1) It prevents the potential risks oftransmission of pathogens from the animal/human feeder cells to thehuman HES cells and the fact that the current system of propagation(human/animal co-culture) has been construed as a xenotransplant; (2) Abetter quality control of HES cells can be reached; (3) A mass/bulkproduction of HES cells is feasible; and (4) It sheds light on clinicalapplication of HES cells.

The examples as disclosed above are used to provide detailed descriptionand are in no way to be considered to limit the scope of the inventionin any manner. All modifications and alterations made by those familiarwith the skill without departing from the spirits of the invention andappended claims shall remain within the protected scope and claims ofthe invention.

1. A culture system for proliferation and undifferentiated growth ofhuman embryonic stem cells comprising: a culture matrix comprisingextracellular matrix prepared from feeder cells; and a conditionedmedium being preconditioned by feeder cells; wherein said feeder cellsare selected from primary mouse embryonic fibroblasts (PMEF), a mouseembryonic fibroblast cell line (MEF), murine fetal fibroblasts (MFF),human embryonic fibroblasts (HEF), human fetal muscle (HFM), human fetalskin cells (HFS), human adult skin cells, human foreskin fibroblasts(HFF), human adult fallopian tubal epithelial cells (HAFT) or humanmarrow stromal cells (hMSCs).
 2. The culture system according to claim1, wherein said feeder cells are mouse embryonic fibroblasts or humanforeskin fibroblasts.
 3. The culture system according to claim 1,wherein said culture matrix is prepared by removing intracellularsubstances by means of treating said feeder cells with NaOH ortrinitrotoluene to obtain extracellular matrix as culture matrix.
 4. Theculture system according to claim 3, wherein the intracellularsubstances are removed by washing with water or buffered solution. 5.The culture system according to claim 4, wherein said buffered solutionis phosphate buffered saline.
 6. The culture system according to claim1, wherein said conditioned medium is prepared by the steps of: (a)inactivating the feeder cells; (b) placing the cells obtained in step(a) in a culture solution; and (c) collecting the cell culture solutionas conditioned medium.
 7. The culture system according to claim 6,wherein said feeder cells are inactivated by irradiation with gamma rayor treatment with mitomycin C.
 8. The culture system according to claim7, wherein said feeder cells are inactivated by treating with mitomycinC.
 9. A method for culturing human embryonic stem cells, comprising thesteps of: obtaining human embryonic stem cells; culturing the humanembryonic stem cells in the culture system described according to claims1; and continuing the culture to maintain human embryonic stem cells ina substantially proliferative and undifferentiated state.
 10. The methodaccording to claim 9, wherein said human embryonic stem cells aremaintained in a substantially undifferentiated and proliferative stateover the course of at least five passages in said culture system.